Solid chimeric organs, animal models having same, process for preparing same, non-tumorigenic immortalized human cell lines, susceptible cells and cytopathic mammalian viruses

Abstract

This invention provides useful solid chimeric organs as well as animal models having such solid chimeric organs and processes for their preparation. Such organs find significant value and use in developing new models for disease, drug and therapeutic investigations and monitoring, and in studying storage functions and processes. The solid chimeric organs are comprised of recipient cells and donor cells, the latter themselves comprising allogeneic or xenogeneic cells which are unmodified or modified to contain one or more nucleic acid segments capable of exhibiting at least one biological property, e.g. DNA synthesis, replication, promoter function, transcription, translation, reverse transcription, and the like, non-native to the donor cell. The solid chimeric organs can be prepared from recipient organs using recipient cells and implanted allogeneic or xenogeneic donor cells from non-homologous organs or tissues. Also provided by this invention are non-tumorigenic immortalized human cell lines modified by having sequences derived from hepatotrophic viruses introduced into the cells. Susceptible cells, cytopathic mammalian viruses, target cells rendered susceptible to cytopathic events, and human cells infected or transfected by noncytopathic viruses are also provided.

What is claimed is:  
     
         1 . A solid chimeric organ comprising: 
 i) recipient cells; and    ii) donor cells comprising allogeneic or xenogeneic cells, said allogeneic or xenogeneic cells modified to contain one or more nucleic acid segments capable of exhibiting at least one biological property non-native to said donor cell.    
     
     
         2 . The solid chimeric organ of    claim 1   , wherein said nucleic acid segments are native or non-native to said donor cells.  
     
     
         3 . The solid chimeric organ of    claim 1   , wherein said nucleic acid segments are produced by or derived from a member selected from the group consisting of one or more nucleic acid constructs, a viral vector, a mutated virus or wild type virus, modified or unmodified oligo- or polynucleotides and a chromosome or chromosomal segment, or a combination of any of the foregoing.  
     
     
         4 . The solid chimeric organ of    claim 1   , wherein said chromosome or chromosomal segment is from a cell taken from a different donor or taken from a cell from a different organ in said recipient.  
     
     
         5 . The solid chimeric organ of    claim 1   , wherein said donor cells are chimeric.  
     
     
         6 . The solid chimeric organ of    claim 5   , wherein said chimeric donor cells are derived from a first and second parental cells which are phenotypically different from each other, and said chimeric donor cells exhibit at least two phenotypic characteristics, at least one of which characteristics is derived from said first parental cell and at least one other characteristic is derived from said second parental cell, and wherein said first or said second parental cells are allogeneic or xenogeneic with respect to said recipient cells.  
     
     
         7 . The solid chimeric organ of    claim 1   , wherein said nucleic acid segment or segments have been introduced into said donor allogeneic or xenogeneic cells prior to the formation of said chimeric organ.  
     
     
         8 . The solid chimeric organ of    claim 1   , wherein said nucleic acid segment or segments have been introduced into said donor allogeneic or xenogeneic cells after the formation of said chimeric organ.  
     
     
         9 . The solid chimeric organ of    claim 1   , wherein the non-native nucleic acid segment has been introduced into said donor allogeneic or xenogeneic cells during the formation of said chimeric organ.  
     
     
         10 . The solid chimeric organ of    claim 1   , wherein said recipient cells are derived from a solid organ capable of regeneration.  
     
     
         11 . The solid chimeric organ of    claim 10   , wherein said solid organ comprises liver or skin.  
     
     
         12 . The solid chimeric organ of    claim 1   , wherein said allogeneic or xenogeneic donor cells are selected from the group consisting of liver, skin and pancreatic islets, or combinations thereof.  
     
     
         13 . The solid chimeric organ of    claim 1   , wherein said biological property is selected from the group consisting of DNA synthesis, DNA replication, promoter function, DNA transcription, DNA translation, reverse transcription, ligation, post-transcriptional modification, transcriptional processing, post-translational processing, protein export, or combinations thereof.  
     
     
         14 . A non-human animal model having the solid chimeric organ of    claim 1   .  
     
     
         15 . The non-human animal model of    claim 14   , wherein said animal was incapable of supporting a viral or pathogenic infection of interest prior to having the solid chimeric organ of    claim 1   , and wherein said animal is rendered capable of supporting said viral infection after having said chimeric organ.  
     
     
         16 . The non-human animal model of    claim 15   , wherein the agent for said viral infection comprises hepatotrophic viruses.  
     
     
         17 . The non-human animal model of    claim 16   , wherein said hepatotrophic viruses are selected from the group consisting of hepatitis A viruses, hepatitis B viruses, hepatitis C viruses, hepatitis D viruses, hepatitis E viruses, or a combination of any of the foregoing.  
     
     
         18 . The non-human animal model of    claim 15   , wherein the agent for said pathogenic infection comprises plasmodium.  
     
     
         19 . A solid chimeric organ prepared from a recipient organ, said solid chimeric organ comprising: 
 (i) recipient cells, and    (ii) implanted allogeneic or xenogeneic donor cells from a non-homologous organ or tissue,    wherein said recipient organ has been reduced in size or the total number of recipient cells in said recipient organ have been reduced prior to or during implantation of said donor cells.    
     
     
         20 . The solid chimeric organ of    claim 19   , further comprising (iii) implanted donor cells from an homologous organ or tissue.  
     
     
         21 . The solid chimeric organ of claims  19  or  20 , wherein said recipient cells (i) are derived from a solid organ capable of regeneration.  
     
     
         22 . The solid chimeric organ of    claim 21   , wherein said solid organ capable of regeneration comprises liver or skin.  
     
     
         23 . The solid chimeric organ of claims  19  or  20 , wherein said allogeneic or xenogeneic donor cells (ii) are selected from the group consisting of liver, skin and pancreatic islets, or combinations thereof.  
     
     
         24 . A process for preparing a solid chimeric organ comprising the steps of: 
 i) removing cells from an organ or tissue of a donor;    ii) introducing into said removed donor cells one or more non-native nucleic acid segments or one or more native nucleic acid segments capable of non-native expression in a target cell, said non-native nucleic acid segments or said native nucleic acid segments being capable of eliciting at least one biological effect non-native to said cell; and    iii) implanting the donor cells into which said segment or segments have been introduced into a desired organ of an allogeneic or xenogeneic host.    
     
     
         25 . A process for preparing a solid chimeric organ comprising the steps of: 
 i) removing cells from an organ or tissue of a donor;    ii) implanting said removed donor cells into a desired organ of an allogeneic or xenogeneic host; and    iii) introducing into said implanted donor cells one or more non-native nucleic acid segments or one or more native nucleic acid segments capable of non-native expression in a target cell, said non-native nucleic acid segment or said native nucleic acid segments being capable of eliciting at least one biological effect non-native to said cell.    
     
     
         26 . The process according to claims  24  or  25 , wherein said introducing and implanting steps are carried out at about the same time.  
     
     
         27 . The process according to    claim 24    or    25   , wherein said introducing step is carried out by a means comprising viral-mediated gene delivery or non-viral mediated gene delivery or both.  
     
     
         28 . The process according to claims  27 , wherein said viral-mediated gene delivery is mediated by an unmodified virus or a virus derivative.  
     
     
         29 . The process according to    claim 28   , wherein said virus or virus derivative is selected from the group consisting of retroviruses, adenoviruses, Adeno-associated viruses (AAV), herpes simplex viruses, Epstein-Barr viruses, hepatotrophic viruses, polioviruses and vaccinia viruses, or a combination of any of the foregoing.  
     
     
         30 . The process according to    claim 29   , wherein said hepatotrophic viruses are selected from the group consisting of hepatitis A viruses, hepatitis B viruses, hepatitis C viruses, hepatitis D viruses, hepatitis E viruses, or a combination of any of the foregoing.  
     
     
         31 . The process according to    claim 27   , wherein said non-viral mediated gene delivery comprises a means selected from the group consisting of calcium phosphate transfection, DEAE-dextran transfection, liposome-mediated transfection, lipofection, electroporation, ligand-mediated delivery, microinjection and PEG-mediated fusion, or combinations thereof.  
     
     
         32 . A process for preparing a solid chimeric organ from a recipient organ which comprises recipient cells, said process comprising the steps of: 
 (a) reducing the size of said recipient organ or reducing the total number of recipient cells in said recipient organ;    (b) providing allogeneic or xenogeneic donor cells from a non-homologous organ or tissue; and    (c) implanting said allogeneic or xenogeneic donor cells provided from step (b) into said recipient organ.    
     
     
         33 . The process of    claim 32   , wherein said providing step (b) or said implanting step (c) or both, further comprises donor cells from a homologous organ or tissue.  
     
     
         34 . The process of claims  32  or  33 , wherein said recipient organ comprises a solid organ capable of regeneration.  
     
     
         35 . The process of    claim 34   , wherein said solid organ capable of regeneration comprises liver or skin.  
     
     
         36 . The process of claims  32  or  33 , wherein said allogeneic or xenogeneic donor cells are selected from the group consisting of liver, skin and pancreatic islets, or combinations thereof.  
     
     
         37 . A non-tumorigenic immortalized human cell line which has been modified by the introduction of a sequence derived from a hepatotrophic virus.  
     
     
         38 . The non-tumorigenic immortalized human cell line of    claim 37   , wherein said hepatotrophic viruses are selected from the group consisting of hepatitis A viruses, hepatitis B viruses, hepatitis C viruses, hepatitis D viruses, hepatitis E viruses, or a combination of any of the foregoing.  
     
     
         39 . The non-tumorigenic immortalized human cell line of    claim 37   , said cell line is capable of producing extracellular particles containing viral nucleic acid or produces extracellular particles containing viral nucleic acid.  
     
     
         40 . The non-tumorigenic immortalized human cell line of    claim 39   , wherein said extracellular particles are capable of producing cellular infection or produce cellular infection.  
     
     
         41 . The non-tumorigenic immortalized human cell line of claim of  39 , wherein said extracellular particles contains one or more sequences not native to hepatitis virus.  
     
     
         42 . A susceptible cell derived from a non-susceptible cell that is a host in vivo for a virus or pathogen of interest, said non-susceptible cell having been modified to render it susceptible to infection by said virus or pathogen of interest, said modification comprising the introduction of one or more native or non-native nucleic acid segments into said cell.  
     
     
         43 . The susceptible cell of    claim 42   , wherein said native or non-native nucleic acid segment or segments are produced by or derived from a member selected from the group consisting of one or more nucleic acid constructs, a viral vector, a mutated virus or wild type virus, modified or unmodified oligo- or polynucleotides and a chromosome or chromosomal segment, or a combination of any of the foregoing.  
     
     
         44 . The animal model of    claim 43   , wherein said non-susceptible cell comprises or is derived from a hepatocyte.  
     
     
         45 . The animal model of    claim 44   , wherein said hepatocyte was grown in vitro to render it non-susceptible.  
     
     
         46 . A non-human animal model having a susceptible cell derived from a non-susceptible cell that has been modified to render it susceptible to a viral or pathogenic infection, said modification having been rendered by implantation.  
     
     
         47 . A susceptible cell derived from a non-susceptible cell that has been modified to render it susceptible to a viral or pathogenic infection, said modification having been rendered by exposure to a cellular component selected from the group consisting of whole cells, cellular membranes, extracellular matrix molecules, and soluble extracts or purified components therefrom, or a combination of any of the foregoing.  
     
     
         48 . The susceptible cell of    claim 47   , wherein said soluble extracts or purified components are selected from the group consisting of cytokines, lymphokines, hormones, immunological factors and growth factors, or a combination of any of the foregoing.  
     
     
         49 . A susceptible cell derived from an animal having cells, said animal being non-susceptible to a virus or pathogen of interest, the cells of said animal having been modified to render such cells susceptible to an infection by said virus or pathogen, said modification comprising the introduction of one or more non-native or native nucleic acid segments into said cells.  
     
     
         50 . An animal model having one or more susceptible cells of    claim 49   .  
     
     
         51 . A cytopathic mammalian virus, said virus being derived from a non-cytopathic virus, said derivation comprising a means selected from the group consisting of a mutation, introduction of additional promoters, and incorporation of other viral nucleic acid sequences, or host cell nucleic acid sequences.  
     
     
         52 . The cytopathic mammalian virus of    claim 51   , wherein said non-cytopathic virus comprises a hepatotrophic virus.  
     
     
         53 . The cytopathic mammalian virus of    claim 52   , wherein said heptotrophic virus comprises a hepadnovirus.  
     
     
         54 . The cytopathic mammalian virus of    claim 53   , wherein said hepadnovirus comprises Hepatitis B Virus.  
     
     
         55 . A target cell which has been rendered susceptible to a cytopathic event upon infection by a noncytopathic virus, said cell having been modified by the introduction of at least one native or non-native nucleic acid construct.  
     
     
         56 . The target cell of    claim 55   , wherein said native or non-native construct is derived from a virus or a cell.  
     
     
         57 . A human cell infected or transfected by a noncytopathic virus, wherein said human cell is rendered susceptible to a cytopathic event by exposing to a biologically active compound selected from the group consisting of a cytokine, a lymphokine, extracellular matrix, a hormone, an apoptosis factor, an immunological factor, or a combination of any of the foregoing.  
     
     
         58 . The human cell of    claim 57   , wherein said cytokine comprises a tumor necrosis factor.  
     
     
         59 . The human cell of    claim 58   , wherein said tumor necrosis factor comprises TNFα.  
     
     
         60 . The human cell of    claim 57   , wherein said apoptosis factor comprises soluble fas ligand.  
     
     
         61 . A process of increasing implantation efficiency comprising the steps of: 
 providing a recipient subject having an organ treated to reduce its mass or volume; and    implanting allogeneic or xenogeneic donor cells into said treated organ, said donor cell having been modified to contain one or more non-native nucleic acid segments.    
     
     
         62 . The process of    claim 61   , wherein said donor cells are taken from an organ of said donor corresponding to said treated organ in said recipient subject.  
     
     
         63 . The process of    claim 61   , wherein said donor cells are taken from an organ of said donor that is different from said treated organ in said recipient.